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Background: MicroRNA (miRNA) play a significant role in the pathogenesis of complex neurodegenerative diseases including age-related macular degeneration (AMD), acting as post-transcriptional gene suppressors through their association with argonaute 2 (AGO2) - a key member of the RNA Induced Silencing Complex (RISC). Identifying the retinal miRNA/mRNA interactions in health and disease will provide important insight into the key pathways miRNA regulate in disease pathogenesis and may lead to potential therapeutic targets to mediate retinal degeneration., Methods: To identify the active miRnome targetome interactions in the healthy and degenerating retina, AGO2 HITS-CLIP was performed using a rodent model of photoreceptor degeneration. Analysis of publicly available single-cell RNA sequencing (scRNAseq) data was performed to identify the cellular location of AGO2 and key members of the microRNA targetome in the retina. AGO2 findings were verified by in situ hybridization (RNA) and immunohistochemistry (protein)., Results: Analysis revealed a similar miRnome between healthy and damaged retinas, however, a shift in the active targetome was observed with an enrichment of miRNA involvement in inflammatory pathways. This shift was further demonstrated by a change in the seed binding regions of miR-124-3p, the most abundant retinal AGO2-bound miRNA, and has known roles in regulating retinal inflammation. Additionally, photoreceptor cluster miR-183/96/182 were all among the most highly abundant miRNA bound to AGO2. Following damage, AGO2 expression was localized to the inner retinal layers and more in the OLM than in healthy retinas, indicating a locational miRNA response to retinal damage., Conclusions: This study provides important insight into the alteration of miRNA regulatory activity that occurs as a response to retinal degeneration and explores the miRNA-mRNA targetome as a consequence of retinal degenerations. Further characterisation of these miRNA/mRNA interactions in the context of the degenerating retina may provide an important insight into the active role these miRNA may play in diseases such as AMD., (© 2021. The Author(s).)

Purpose: Dysregulation of the complement cascade contributes to a variety of retinal dystrophies, including age-related macular degeneration (AMD). The central component of complement, C3, is expressed in abundance by macrophages in the outer retina, and its ablation suppresses photoreceptor death in experimental photo-oxidative damage. Whether this also influences macrophage reactivity in this model system, however, is unknown. We investigate the effect of C3 ablation on macrophage activity and phagocytosis by outer retinal macrophages during photo-oxidative damage., Methods: Age-matched C3 knockout (KO) mice and wild-type (WT) C57/Bl6 mice were subjected to photo-oxidative damage. Measurements of the outer nuclear layer (ONL) thickness and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were used to assess pathology and photoreceptor apoptosis, respectively. Macrophage abundance and phagocytosis were assessed with immunolabeling for pan-macrophage and phagocytic markers, in conjunction with TUNEL staining in cohorts of C3 KO and WT mice., Results: The C3 KO mice exhibited protection against photoreceptor cell death following photo-oxidative damage, which was associated with a reduction in immunoreactivity for the stress-related factor GFAP. In conjunction, there was a reduction in IBA1-positive macrophages in the outer retina compared to the WT mice and a decrease in the number of CD68-positive cells in the outer nuclear layer and the subretinal space. In addition, the engulfment of TUNEL-positive and -negative photoreceptors by macrophages was significantly lower in the C3 KO mice cohort following photo-oxidative damage compared to the WT cohort., Conclusions: The results show that the absence of C3 mitigates the phagocytosis of photoreceptors by macrophages in the outer retina, and the net impact of C3 depletion is neuroprotective in the context of photo-oxidative damage. These data improve our understanding of the impact of C3 inhibition in subretinal inflammation and inform the development of treatments for targeting complement activation in diseases such as AMD., (Copyright © 2020 Molecular Vision.)

Purpose: The use of small non-coding nucleic acids, such as siRNA and miRNA, has allowed for a deeper understanding of gene functions, as well as for development of gene therapies for complex neurodegenerative diseases, including retinal degeneration. For effective delivery into the eye and transfection of the retina, suitable transfection methods are required. We investigated the use of a lipid-based transfection agent, Invivofectamine ® 3.0 (Thermo Fisher Scientific), as a potential method for delivery of nucleic acids to the retina., Methods: Rodents were injected intravitreally with formulations of Invivofectamine 3.0 containing scrambled, Gapdh, Il-1β , and C3 siRNAs, or sterile PBS (control) using a modified protocol for encapsulation of nucleic acids. TdT-mediated dUTP nick-end labeling (TUNEL) and IBA1 immunohistochemistry was used to determine histological cell death and inflammation. qPCR were used to determine the stress and inflammatory profile of the retina. Electroretinography (ERG) and optical coherence tomography (OCT) were employed as clinical indicators of retinal health., Results: We showed that macrophage recruitment, retinal stress, and photoreceptor cell death in animals receiving Invivofectamine 3.0 were comparable to those in negative controls. Following delivery of Invivofectamine 3.0 alone, no statistically significant changes in expression were found in a suite of inflammatory and stress genes, and ERG and OCT analyses revealed no changes in retinal function or morphology. Injections with siRNAs for proinflammatory genes ( C3 and Il-1β ) and Gapdh , in combination with Invivofectamine 3.0, resulted in statistically significant targeted gene knockdown in the retina for up to 4 days following injection. Using a fluorescent Block-It siRNA, transfection was visualized throughout the neural retina with evidence of transfection observed in cells of the ganglion cell layer, inner nuclear layer, and outer nuclear layer., Conclusions: This work supports the use of Invivofectamine 3.0 as a transfection agent for effective delivery of nucleic acids to the retina for gene function studies and as potential therapeutics., (Copyright © 2020 Molecular Vision.)

Purpose: We sought to determine the role and retinal cellular location of microRNA-124 (miR-124) in a neuroinflammatory model of retinal degeneration. Further, we explored the anti-inflammatory relationship of miR-124 with a predicted messenger RNA (mRNA) binding partner, chemokine (C-C motif) ligand 2 (Ccl2), which is crucially involved in inflammatory cell recruitment in the damaged retina., Methods: Human AMD donor eyes and photo-oxidative damaged (PD) mice were labeled for miR-124 expression using in situ hybridization. PDGFRa-cre RFP mice were used for Müller cell isolation from whole retinas. MIO-M1 immortalized cells and rat primary Müller cells were used for in vitro analysis of miR-124 expression and its relationship with Ccl2. Therapeutic efficacy was tested with intravitreal administration of miR-124 mimic in mice, with electroretinography used to determine retinal function. IBA1 immunohistochemistry and photoreceptor row counts were used for assessment of inflammation and cell death., Results: MiR-124 expression was correlated with progressive retinal damage, inflammation, and cell death in human AMD and PD mice. In addition, miR-124 expression was inversely correlated to Ccl2 expression in mice following PD. MiR-124 was localized to both neuronal-like photoreceptors and glial (Müller) cells in the retina, with a redistribution from neurons to glia occurring as a consequence of PD. Finally, intravitreal administration of miR-124 mimics decreased retinal inflammation and photoreceptor cell death, and improved retinal function., Conclusions: This study has provided an understanding of the mechanism behind miR-124 in the degenerating retina and demonstrates the usefulness of miR-124 mimics for the modulation of retinal degenerations.

Purpose: Systemic increases in reactive oxygen species, and their association with inflammation, have been proposed as an underlying mechanism linking obesity and age-related macular degeneration (AMD). Studies have found increased levels of oxidative stress biomarkers and inflammatory cytokines in obese individuals; however, the correlation between obesity and retinal inflammation has yet to be assessed. We used the leptin-deficient (ob/ob) mouse to further our understanding of the contribution of obesity to retinal oxidative stress and inflammation., Methods: Retinas from ob/ob mice were compared to age-matched wild-type controls for retinal function (electroretinography) and gene expression analysis of retinal stress ( Gfap ), oxidative stress ( Gpx3 and Hmox1 ), and complement activation ( C3 , C2 , Cfb , and Cfh ). Oxidative stress was further quantified using a reactive oxygen species and reactive nitrogen species (ROS and RNS) assay. Retinal microglia and macrophage migration to the outer retina and complement activation were determined using immunohistochemistry for IBA1 and C3, respectively. Retinas and sera were used for metabolomic analysis using QTRAP mass spectrometry., Results: Retinal function was reduced in ob/ob mice, which correlated to changes in markers of retinal stress, oxidative stress, and inflammation. An increase in C3-expressing microglia and macrophages was detected in the outer retinas of the ob/ob mice, while gene expression studies showed increases in the complement activators ( C2 and Cfb ) and a decrease in a complement regulator ( Cfh ). The expression of several metabolites were altered in the ob/ob mice compared to the controls, with changes in polyunsaturated fatty acids (PUFAs) and branched-chain amino acids (BCAAs) detected., Conclusions: The results of this study indicate that oxidative stress, inflammation, complement activation, and lipid metabolites in the retinal environment are linked with obesity in ob/ob animals. Understanding the interplay between these components in the retina in obesity will help inform risk factor analysis for acquired retinal degenerations, including AMD.

Purpose: Complement system dysregulation is strongly linked to the progression of age-related macular degeneration (AMD). Deposition of complement including C3 within the lesions in atrophic AMD is thought to contribute to lesion growth, although the contribution of local cellular sources remains unclear. We investigated the role of retinal microglia and macrophages in complement activation within atrophic lesions, in AMD and in models of focal retinal degeneration., Methods: Human AMD donor retinas were labeled for C3 expression via in situ hybridization. Rats were subject to photo-oxidative damage, and lesion expansion was tracked over a 2-month period using optical coherence tomography (OCT). Three strategies were used to determine the contribution of local and systemic C3 in mice: total C3 genetic ablation, local C3 inhibition using intravitreally injected small interfering RNA (siRNA), and depletion of serum C3 using cobra venom factor., Results: Retinal C3 was expressed by microglia/macrophages located in the outer retina in AMD eyes. In rodent photo-oxidative damage, C3-expressing microglia/macrophages and complement activation were located in regions of lesion expansion in the outer retina over 2 months. Total genetic ablation of C3 ameliorated degeneration and complement activation in retinas following damage, although systemic depletion of serum complement had no effect. In contrast, local suppression of C3 expression using siRNA inhibited complement activation and deposition, and reduced cell death., Conclusions: These findings implicate C3, produced locally by retinal microglia/macrophages, as contributing causally to retinal degeneration. Consequently, this suggests that C3-targeted gene therapy may prove valuable in slowing the progression of AMD.

Background: The recruitment of macrophages accompanies almost every pathogenic state of the retina, and their excessive activation in the subretinal space is thought to contribute to the progression of diseases including age-related macular degeneration. Previously, we have shown that macrophages aggregate in the outer retina following damage elicited by photo-oxidative stress, and that inhibition of their recruitment reduces photoreceptor death. Here, we look for functional insight into macrophage activity in this model through the spatiotemporal interplay of macrophage polarisation over the course of degeneration., Methods: Rats were exposed to 1000 lux light damage (LD) for 24 hrs, with some left to recover for 3 and 7 days post-exposure. Expression and localisation of M1- and M2- macrophage markers was investigated in light-damaged retinas using qPCR, ELISA, flow cytometry, and immunohistochemistry., Results: Expression of M1- (Ccl3, Il-6, Il-12, Il-1β, TNFα) and M2- (CD206, Arg1, Igf1, Lyve1, Clec7a) related markers followed discrete profiles following light damage; up-regulation of M1 genes peaked at the early phase of cell death, while M2 genes generally exhibited more prolonged increases during the chronic phase. Moreover, Il-1β and CD206 labelled accumulations of microglia/macrophages which differed in their morphological, temporal, and spatial characteristics following light damage., Conclusions: The data illustrate a dynamic shift in macrophage polarisation following light damage through a broad swathe of M1 and M2 markers. Pro-inflammatory M1 activation appears to dominate the early phase of degeneration while M2 responses appear to more heavily mark the chronic post-exposure period. While M1/M2 polarisation represents two extremes amongst a spectrum of macrophage activity, knowledge of their predominance offers insight into functional consequences of macrophage activity over the course of damage, which may inform the spatiotemporal employment of therapeutics in retinal disease.

Resident microglial cells can be regarded as the immunological watchdogs of the brain and the retina. They are active sensors of their neuronal microenvironment and rapidly respond to various insults with a morphological and functional transformation into reactive phagocytes. There is strong evidence from animal models and in situ analyses of human tissue that microglial reactivity is a common hallmark of various retinal degenerative and inflammatory diseases. These include rare hereditary retinopathies such as retinitis pigmentosa and X-linked juvenile retinoschisis but also comprise more common multifactorial retinal diseases such as age-related macular degeneration, diabetic retinopathy, glaucoma, and uveitis as well as neurological disorders with ocular manifestation. In this review, we describe how microglial function is kept in balance under normal conditions by cross-talk with other retinal cells and summarize how microglia respond to different forms of retinal injury. In addition, we present the concept that microglia play a key role in local regulation of complement in the retina and specify aspects of microglial aging relevant for chronic inflammatory processes in the retina. We conclude that this resident immune cell of the retina cannot be simply regarded as bystander of disease but may instead be a potential therapeutic target to be modulated in the treatment of degenerative and inflammatory diseases of the retina., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Introduction: Complement activation is thought to contribute to the pathogenesis of age-related macular degeneration (AMD), which may be mediated in part by para-inflammatory processes. We aimed to investigate the expression and localization of C3, a crucial component of the complement system, in the retina during the course of aging., Methods: SD rats were born and reared in low-light conditions, and euthanized at post-natal (P) days 100, 450, or 750. Expression of C3, IBA1, and Ccl- and Cxcl- chemokines was assessed by qPCR, and in situ hybridization. Thickness of the ONL was assessed in retinal sections as a measure of photoreceptor loss, and counts were made of C3-expressing monocytes., Results: C3 expression increased significantly at P750, and correlated with thinning of the ONL, at P750, and up-regulation of GFAP. In situ hybridization showed that C3 was expressed by microglia/monocytes, mainly from within the retinal vasculature, and occasionally the ONL. The number of C3-expressing microglia increased significantly by P750, and coincided spatiotemporally with thinning of the ONL, and up-regulation of Ccl- and Cxcl- chemokines., Conclusions: Our data suggest that recruited microglia/monocytes contribute to activation of complement in the aging retina, through local expression of C3 mRNA. C3 expression coincides with age-related thinning of the ONL at P750, although it is unclear whether the C3-expressing monocytes are a cause or consequence. These findings provide evidence of activation of complement during natural aging, and may have relevance to cellular events underling the pathogenesis of age-related retinal diseases.

Introduction: To investigate the validity of using 670nm red light as a preventative treatment for Retinopathy of Prematurity in two animal models of oxygen-induced retinopathy (OIR)., Materials and Methods: During and post exposure to hyperoxia, C57BL/6J mice or Sprague-Dawley rats were exposed to 670 nm light for 3 minutes a day (9J/cm²). Whole mounted retinas were investigated for evidence of vascular abnormalities, while sections of neural retina were used to quantify levels of cell death using the TUNEL technique. Organs were removed, weighed and independent histopathology examination performed., Results: 670 nm light reduced neovascularisation, vaso-obliteration and abnormal peripheral branching patterns of retinal vessels in OIR. The neural retina was also protected against OIR by 670 nm light exposure. OIR-exposed animals had severe lung pathology, including haemorrhage and oedema, that was significantly reduced in 670 nm+OIR light-exposed animals. There were no significance differences in the organ weights of animals in the 670 nm light-exposed animals, and no adverse effects of exposure to 670 nm light were detected., Discussion: Low levels of exposure to 670 nm light protects against OIR and lung damage associated with exposure to high levels of oxygen, and may prove to be a non-invasive and inexpensive preventative treatment for ROP and chronic lung disease associated with prematurity.

Aim: Complement activation is associated with the pathogenesis of age-related macular degeneration (AMD). We aimed to investigate whether 670-nm light treatment reduces the propagation of complement in a light-induced model of atrophic AMD., Methods: Sprague-Dawley (SD) rats were pretreated with 9 J/cm(2) 670-nm light for 3 minutes daily over 5 days; other animals were sham treated. Animals were exposed to white light (1,000 lux) for 24 h, after which animals were kept in dim light (5 lux) for 7 days. Expression of complement genes was assessed by quantitative polymerase chain reaction (qPCR), and immunohistochemistry. Counts were made of C3-expressing monocytes/microglia using in situ hybridization. Photoreceptor death was also assessed using outer nuclear layer (ONL) thickness measurements, and oxidative stress using immunohistochemistry for 4-hydroxynonenal (4-HNE)., Results: Following light damage, retinas pretreated with 670-nm light had reduced immunoreactivity for the oxidative damage maker 4-HNE in the ONL and outer segments, compared to controls. In conjunction, there was significant reduction in retinal expression of complement genes C1s, C2, C3, C4b, C3aR1, and C5r1 following 670 nm treatment. In situ hybridization, coupled with immunoreactivity for the marker ionized calcium binding adaptor molecule 1 (IBA1), revealed that C3 is expressed by infiltrating microglia/monocytes in subretinal space following light damage, which were significantly reduced in number after 670 nm treatment. Additionally, immunohistochemistry for C3 revealed a decrease in C3 deposition in the ONL following 670 nm treatment., Conclusions: Our data indicate that 670-nm light pretreatment reduces lipid peroxidation and complement propagation in the degenerating retina. These findings have relevance to the cellular events of complement activation underling the pathogenesis of AMD, and highlight the potential of 670-nm light as a non-invasive anti-inflammatory therapy.

Purpose: To investigate the expression and localization of complement system mRNA and protein in a light-induced model of progressive retinal degeneration., Methods: Sprague-Dawley (SD) rats were exposed to 1000 lux of bright continuous light (BCL) for up to 24 hours. At time points during (1-24 hours) and after (3 and 7 days) exposure, the animals were euthanatized and the retinas processed. Differential expression of complement genes at 24 hours of exposure was assessed using microarray analysis. Expression of complement genes was validated by quantitative PCR, and expression of selected genes was investigated during and after BCL exposure. Photoreceptor apoptosis was assessed using TUNEL and C3 was further investigated by spatiotemporal analysis using in situ hybridization and immunohistochemistry., Results: Exposure to 24 hours of BCL induced differential expression of a suite of complement system genes, including classic and lectin components, regulators, and receptors. C1qr1, MCP, Daf1, and C1qTNF6 all modulated in concert with photoreceptor death and AP-1 expression, which reached a peak at 24 hours exposure. C1s and C4a reached peak expression at 3 days after exposure, while expression of C3, C3ar1, and C5r1 were maximum at 7 days after exposure. C3 mRNA was detected in ED1- and IBA1-positive microglia/macrophages, in the retinal vessels and optic nerve head and in the subretinal space, particularly at the margins of the emerging lesion., Conclusions: The data indicate that BCL induces the prolonged expression of a range of complement genes and show that microglia/macrophages synthesize C3 and deposit it in the ONL after BCL injury. These findings have relevance to the role of complement in progressive retinal degeneration, including atrophic AMD.

Purpose: To investigate the time course and localization of Ccl2 expression and recruitment of inflammatory cells associated with light-induced photoreceptor degeneration., Methods: Sprague-Dawley (SD) rats were exposed to 1000 lux light for up to 24 hours, after which some animals were allowed to recover in dim light (5 lux) for 3 or 7 days. During and after exposure to light, the animals were euthanatized and the retinas processed. Ccl2 expression was assessed by qPCR, immunohistochemistry, and in situ hybridization at each time point. Counts were made of perivascular monocytes/microglia immunolabeled with ED1, and photoreceptor apoptosis was assessed with TUNEL., Results: Upregulation of Ccl2 expression was evident in the retina by 12 hours of exposure and correlated with increased photoreceptor death. Ccl2 expression reached its maximum at 24 hours, coinciding with peak cell death. Immunohistochemistry and in situ hybridization showed that Ccl2 is expressed by Müller cells from 12 hours of exposure, most intensely in the superior retina, in the region of the incipient light-induced lesion. After the Müller cell-driven expression of Ccl2, there was a substantial recruitment of monocytes to the local retina and choroidal vasculature. This coincided spatially with the expression of Ccl2 in the superior retina. Peak monocyte infiltration followed maximum Ccl2 expression by up to 3 days. Furthermore, Ccl2 immunoreactivity was observed in many infiltrating monocytes after a 24-hour exposure., Conclusions: The data indicate that photoreceptor death promotes region-specific expression of Ccl2 by Müller cells, which facilitates targeting of monocytes to sites of injury. The data suggest that recruitment of monocytes to developing lesions is secondary to signaling events in the retina., (Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc.)